The pentahydric alcohol xylitol is a sugar alcohol, with significant application in food and confectionary industry as low calorie sweetener. It is widely distributed throughout nature in small amounts, with some of the best sources being fruits, vegetables, berries, mushrooms, lettuce, hardwoods, and corncobs. Human body produces 5-10 gms of xylitol from food source using established energy pathways. Although widely distributed in nature, its presence in low concentration makes it uneconomic to produce xylitol on commercial scale from such natural sources.
Xylitol has numerous advantages including the same sweetness as sucrose but with one-third fewer calories and no unpleasant aftertaste. After addition its negative heat of dissolution imparts a cool and refreshing sensation in the oral cavity, making it a popular sweetener for candies and sweets. Xylitol is cariostatic and even a non-carcinogenic substance. Xylitol finds favor with diabetic patients as it is metabolized independently from insulin in the human body (J. WEI, Q. YUAN, T WANG, Le WANG, Front. Chem. Eng. China 2010, 4(1):57-64). It has lower glycemic index value of 13 as against glucose with 100. Xylitol has also been shown to have therapeutic properties and reportedly builds immunity, fights against chronic degenerative diseases, is anti-aging, and has no known toxic levels.
The present invention relates to the microbial production of xylitol, from wood xylose as an enriched fraction of pentose sugar obtained from biomass, by an unexplored wild type Candida strain. More particularly, the invention relates to a novel process developed using a wild strain of Candida tropicalis for xylitol production with high yield and productivity. The invention relates to a process that uses wild type strain of Candida tropicalis, wherein the use of wild type strain always favors over genetically modified one from regulation point of view and overcomes issues related for the product being labeled under food category norms. The present invention also explores Candida tropicalis (NRRL 12968) to an extent which is capable of fermenting pentose sugar at higher rate and that can be achieved using different strains of same species currently known to the art.

The importance of xylitol and its application in different industries require methods for maximum production of this sugar alcohol in an efficient manner. As xylitol is present in low concentrations in vegetables or fruits, its extraction is uneconomical from these sources. Thus xylitol can be produced via the direct reduction of xylose sugar in presence of suitable reducing agents using two main methods: direct reduction by synthetic chemical method and by natural microbial route.
Commercially xylitol is produced by chemical reduction of xylose, a hydrolysate fraction of hemicellulose present in woods, rice straw, millet, etc. An example of this process is described in the U.S. Pat. No. 4,008,285, in which the production of xylitol on a commercial scale is carried out by acid hydrolysis of pentosan-containing raw materials such as wood, corncobs, straw, bran, and cottonseed hulls. The hydrolysis of xylitol is usually carried out using Raney nickel catalyst (NiZAl2O3) at high temperature (80-140° C.) and pressure (up to 50 atm). One limitation of the chemical process is the difficulty of separation and purification of xylose or xylitol from hydrolysates containing other polymer sugars derived from hemicellulose fractions (Jeffries T. W., Kurtzman C. P., Enzyme Microbial Technology, November 1994, Vol. 16, Issue 11: 922-932). Multistep separation techniques, including mechanical filtration and chromatography are required to obtain pure xylitol. These processes adversely affect the cost of production for a yield in the range of 50-60%. Furthermore, such processes involve high temperature and high-pressure associated risks. Waste disposal due to use of acid or alkali is another major concern that is associated with chemical production of xylitol (E. Winkelhausen and S. Kuzmanova, Journal of fermentation and bioengineering, 1998, Vol. 86, no. 1, 1-14). These factors make the chemical methods for the routine production of xylitol difficult, expensive and inefficient.
Recently microbial production of xylitol is explored in literature which offers cost effective downstream processing that can reduce manufacturing cost (Rivas B. et al, Enzyme Microb. Technol., 2003, 31:431-438). Such process would reduce the need for purified xylose, producing high pure, easy to separate product, and be adaptable to wide variety of raw material source from different geographical locations.
Most of the prior art methods reported in the literature shows application of yeast as biochemical catalyst for xylitol production because they are considered to be the best xylitol producers among microorganisms. Screening of more than 30 yeast strains revealed that yeast from genus Candida, such as C. guilliermondii VTT-C-71006, C. tropicalis ATCC 1369 and C. tropicalis ATCC 9968 are best xylitol producers (Ojamo, H, Ph.D. Thesis Helsinki University of technology, Espoo, Finland, 1994).
One of such methods described in prior art, using xylose fermenting yeast Candida tropicalis ATCC 13803 uses initial xylose concentration of 15% (PCT/IN2009/000027). The reported method discloses xylose fermentation in water solution with 50% of sugar conversion and 50% of yield in 196 hrs. Similar work has been reported utilizing Candida tropicalis ATCC 13803 wherein the initial Xylose concentration reported was 10% which is at lower side as compared to the earlier report (Patent No. KR100259470).
Another method reported in prior art used Candida tropicalis ATCC 9968 which has utilized higher initial xylose concentration of 5%-30% than previous reported processes (PCT/FI1990/000015).
Patent No. KR100199819 describes the use of Candida tropicalis KFCC 10960 with initial xylose concentration of 5-12%. One of prior art method reported 173 g/l of xylitol production from initial 200 g/l of xylose concentration in 120 hrs of fermentation using indigenously isolated yeast strain of genus Candida (T. Ikeuchi, M. Azuma, J. Kato, H Ooshima, Biomass and Bioenergy, 1999, 16, 333-339).
One of such methods described in prior art, using xylose fermenting yeast Candida tropicalis ATCC 750 (equivalent to NRRL 12968) yields less than 50% of xylitol from xylose mixture obtained from biomass hydrolysate (Thomas P. West, World J. Microbiol. Biotechnol, 2009, 25:913-916). The invention reports maximum product yield of 0.43 (gm of xylitol/gm of xylose) in 120 hrs of fermentation.
One of such methods described in prior art, using xylose fermenting yeast Candida tropicalis ATCC 7349 reports very low xylose concentration in the fermentation broth with lower xylitol yield. The initial xylose concentration used in the prior art process was 30 g/liter and yields less than 40% of xylitol from xylose mixture obtained from biomass hydrolysis (SAROTE S, MICHAEL S, MANFRED R, Journal of Fermentation and Bioengeering, 1995, Vol. 80, No. 6, 565-570).
Despite significant amount of prior art work, development of commercially feasible microbial production process has remained elusive for number of reasons. Those prior art methods reported in literature had either one or more of different drawbacks such as lower initial substrate concentration, poor conversion and molar/gram yield, and overall process productivity which do not reach the levels necessary for a commercial process. Thus the present invention describes a novel, cost efficient, and high yielding process, wherein higher xylose concentration can be used for commercial xylitol production at better economics.